Overall, the expertise of the Mass Spectrometry Unit is being widely used to further the research of multiple groups within the CCR. In FY2015, the unit collaborated in 40 different projects, with more than 1700 samples processed and analyzed. These projects are being performed in collaboration with 27 different CCR investigators. Among these are projects to characterize the post-translational modifications of target proteins, including sites of phosphorylation, ubiquitination, acetylation, and methylation, better understand signal transduction and protein regulation. The resource is also being used to identify protein interactors of both proteins and nucleic acids, including identification of those that change following post-translational modification. These studies help provide critical insight into protein function and regulation. Mass spectrometry is being used extensively for large-scale quantitative proteomics projects. In these, labeled or label-free methods are used to comprehensively identify whole proteomics, such as the protein composition of conditioned media, biological fluids (in particular, suction blister fluid), a subcellular organelle or vesicle (i.e., mitochondria and exosomes), or a whole cell. Additionally, we are collaborating on two global quantitative phosphoproteome studies, in which the global level of phosphorylation is compared. These discovery-oriented studies, which are sample- and time-intensive, provide broad information for defining new hypotheses and provide new insight into global protein activities and cellular responses. Finally, we are collaborating on two projects that make use of targeted quantitation to compare the protein level of particular proteins in complex samples; these studies are used for quantitation of a small number of specific target proteins in a large number of clinical samples to help validate potential biomarkers. The Mass Spectrometry Unit is also adding hydrogen-deuterium exchange mass spectrometry (HDX-MS) to the techniques available to CCR collaborators. In an HDX-MS experiment, the protein of interest is incubated with a solution of deuterium for varying times, allowing the surface-accessible protons to exchange with deuterium in the solvent. Exchange is quenched by treatment with acid, then the protein is digested with pepsin and the peptides analyzed by mass spectrometry. By analyzing the change in deuterium uptake over time, protein dynamics and conformational changes can be probed. Furthermore, by comparing the exchange profile of a protein in different conditions or in the presence of a binding partner or ligand, more detailed information about changes in the protein conformation can be obtained. For these experiments, we have a robotic liquid handling apparatus that allows automation of the sample preparation steps, from deuterium exchange reaction to injection onto the mass spectrometer, and provides temperature control which is critical to minimize back-exchange of the deuterium to hydrogen. We are also purchasing a Q Exactive Plus mass spectrometer to be primarily dedicated to these experiments. In the past year, two collaborative studies have been published. In addition, several other projects are nearing completion and will be prepared as manuscripts for publication. The first study, published in Nature Communications, is the result of an on-going collaboration with Dr. Curtis Harris, Laboratory of Human Carcinogenesis, to study the biological regulation of the delta133 isoform of p53. Using cell-based methods, we demonstrated that indeed delta133p53 was degraded by selective autophagy, and mass spectrometry experiments identified two ubiquitinated lysine residues in the C-terminal regulatory domain that were responsible for this effect. Further, mass spectrometric identification of interacting proteins indicated that delta133p53 forms a complex with the Hsp70 chaperone complex and the STUB1 E3 ubiquitin ligase. Mutation of the identified ubiquitination sites did reduce the level of delta133p53, but it did not abolish it. Thus, we are continuing to characterize the post-translational modification of delta133 p53 to look for other sites of ubiquitination. In addition, we are looking at additional protein interactors of this isoform of p53 to better understand its biological functions. In a second study, we collaborated with Drs. Ettore Appella, Laboratory of Cell Biology, and Chu-Xia Deng, formerly of NIDDK and currently at the University of Macau, SAR of People's Republic of China. Mass spectrometry was used to investigate the interactors of SIRT1, a histone deacetylase involved in numerous biological functions. Previously, Dr. Deng had demonstrated that SIRT1 deficiency resulted in genome instability, leading to the development of tumors, but the precise mechanisms for this phenotype was unknown. Among the protein interactors of SIRT1 identified were several proteins involved in the DNA replication, such as TopBP1, which plays an essential role in DNA replication fork protection and replication origin firing. Biological assays demonstrated that loss of SIRT1 resulted in increased replication origin firing, asymmetric fork progression, defective intra-S-phase checkpoint, and chromosome damage. In addition, characterization of the sites of acetylation of TopBP1 identified seven modified lysine residues, some of which were deacetylated by SIRT1. Thus, we were able to show that SIRT1 deficiency results in increased acetylation of TopBP1, which in turn causes repression of replication origin firing, indicating that SIRT1 acts upstream of TopBP1 to maintaining genome stability by modulating DNA replication fork initiation and the intra-S-phase cell cycle checkpoint. This research was published in the International Journal of Biological Sciences.